PROJECT SUMMARY Leptin resistance (LR) is a hallmark in obesity. Leptin is an adipokine hormone that exerts its anorexigenic effect through its cognate receptor, the leptin receptor long isoform b (LepRb) to control food intake, body weight and energy expenditure. Therefore LepRb has emerged as a key player in the neuroendocrine control of energy homeostasis. Protein glycosylation is an underappreciated posttranslational modification that controls protein folding, stability and function. N-glycosylation regulates protein trafficking, plasma membrane retention time and dimerization. Additionally O-GlcNAcylation regulates activity and signal transduction of cell surface receptors. Despite this knowledge, very little is known about LepRb glycosylation and its role in the regulation of leptin sensitivity. Glycans also function homeostatically within the gut in conjunction with carbohydrate-binding proteins (lectins) to establish and maintain healthy gut physiology and gut microbes; reciprocally, gut microbes influence host glycosylation. Robust evidence implicates the gut microbiota as an important mediator of obesity. In diet-induced obesity, consumption of a high fat diet (HFD) leads to an altered microbiota (dysbiosis), increased blood levels of bacterial lipopolysaccharide (LPS) and increased secretion of host lectins (i.e. Galectin 3 [Gal3]). Our previous studies show that LPS induces LR in vagal afferent neurons (VAN), via an unknown mechanism. Our unpublished data show that LPS alters the cell surface N- glycome of hypothalamic cells in vivo and in vitro, and decreases phosphorylation of STAT3, a key effector of the leptin signaling pathway. The overarching goal of this proposal will be to identify and determine the mechanism of action by which gut microbes metabolites influence LepRb glycosylation leading to development of leptin resistance in diet induced obesity. The specific aims are: 1) Determine whether HFD-induce dysbiosis drives LR by altering LepRb N-glycosylation and O-GlcNAc modification; receptor cellular localization and signaling in hypothalamus and VAN. 2) Determine the role of LPS inducing the expression of N- glycans with free terminal galactose residues in LepRb, allowing Gal3 to bind to LepRb N- glycans, impairing downstream signaling. A key component of this proposal is the use of the state of the art mass spectrometry technologies to interrogate the influence of the gut microbiota on the leptin receptor glycosylation and signaling. The expected outcomes are to obtain a detailed characterization of N-glyans and O-GlcNAc modification on LepRb in normal and diet induced obesity conditions and to demonstrate the role of LPS and Gal3 on impairing LepRb glycosylation and signaling; thereby identifying novel molecular targets to treat obesity.